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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 16 January 2014 and 10 April 2014. The in-life phase of the study was conducted between 19 January 2014 (first day of treatment) and 06 February 2014 (final day of necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A positive result was seen in the CHO HPRT forward mutation assay and a toxicokinetic assessment based on available data showed limited evidence of absorption of the test substance in view of this a pre-natal developmental toxicity study was conducted to investigate the substances effect on developmental toxicity and address concerns for worker safety. The study also fills an Annex IX REACH data gap.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 194 to 272g.The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were no deviations from these targets. The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on exposure:
Test Item Formulation and Experimental Preparation:For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in distilled water.The stability and homogeneity of the test item formulations was previously determined and results showed the formulations to be stable for at least seven days. Formulations were prepared on four occasions and stored at approximately +4 °C in the dark.Procedure:Four dose groups were allocated:Control: 0 mg/kg bw/day (active ingredient)Low: 100 mg/kg bw/day (active ingredient)Intermediate: 500 mg/kg bw/day (active ingredient)High: 1000 mg/kg bw/day (active ingredient)See any other information on materials and methods section for furtehr details on treatment groups. The test item was administered daily, from Day 5 to Day 19 of gestation, by gavage using a Stainless Steel dosing cannula attached to a graduated Syringe. The volume of test and control item administered to each animal was based on the most recent scheduled body weight. Control animals were treated in an identical manner with the vehicle alone (Distilled water).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of Polyphosphoric acids,esters, with triethanolamine, sodium salts.The test item concentration in the test samples was determined by inductively coupled plasma with mass spectrometric detection (ICP-MS) using an external standard technique. The element analysed was phosphorous. The formulations investigated during the study were found to comprise test item in the range of 100% to 106% and the required content limit of ±10% with reference to the nominal content was met.The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method was validated and proven to be suitable for use.
Details on mating procedure:
The female animals were mated and pregnant prior to dose administration.
Duration of treatment / exposure:
From Day 5 to Day 19 of gestation
Frequency of treatment:
Daily
Duration of test:
All females were terminated on Day 20 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
act. ingr.
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
act. ingr.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
act. ingr.
No. of animals per sex per dose:
24 females per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on previous toxicity data, including a rat OECD 422 toxicity Study.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS:Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.BODY WEIGHT:Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).FOOD CONSUMPTION:Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.WATER CONSUMPTION:Water intake was observed daily by visual inspection of the water bottles for any overt changes.POST MORTEM:All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes The ovaries and uteri of pregnant females were removed, examined and the following data recorded:i) Number of corpora luteaii) Number, position and type of intrauterine implantationiii) Fetal sexiv) External fetal appearancev) Fetal weightvi) Placental weightvii) Gravid uterus weightThe uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.Implantation types were divided into:Early Death/Resorption: No visible distinction between placental/decidual tissue and embryonic tissueLate Death/ Resorption: Separate embryonic/fetal and placental tissue visibleDead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposesAll implantations and viable fetuses were numbered according to their intrauterine position.
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and visceral alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
Data was assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test.Probability values (p) are presented as follows:p<0.01 **p<0.05 *p≥0.05 (not significant)
Indices:
Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. As the litter was standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.Pre and Post Implantation Loss:Percentage pre-implantation loss was calculated as: (number of corpora lutea - number of implantations / number of corpora lutea) x 100Percentage post-implantation loss was calculated as:(number of implantations - number of live fetuses / number of implantations) x 100Sex ratio:Sex ratio was calculated as:% male fetuses (sex ratio) = Number of male fetuses / Total number of fetuses x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Please see attached background material for data tables.Females treated with 1000 and 500 mg/kg bw/day showed a statistically significant increase in body weight gain between Days 5 and 6 and Days 5 and 11 (respectively). An increase in body weight gain is not considered to represent an adverse effect of treatment. Females treated with 1000 and 500 mg/kg bw/day also showed a statistically significant reduction in body weight gain between Days 11 and 14. A true dose related response was not evident and no effect was apparent on overall body weight development therefore the intergroup differences were considered of no toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Dead fetuses:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
gross pathology
maternal abnormalities
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Please see attached background material for data tables.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Please see attached background material for data tables.One fetus from a female treated with 1000 mg/kg bw/day was small and had a cleft lip, shortened upper jaw, malpositioned pinnae, bulging eyes and a cleft palate. Whilst these observations are considered to represent malformation of the fetus, in isolation and without any other treatment-related findings the intergroup difference was considered to be a spontaneous event and not related to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Please see attached background material for data tables.Females treated with 1000 and 500 mg/kg bw/day showed a statistically significant increase in the percent of fetuses showing ossified odontoid. Females treated with 500 and 100 mg/kg bw/day also showed a statistically significant increase in the percent of fetuses showing ossification of the ventral arch of vertebra 1. The observations of isolated variants at a higher incidence compared with controls is not significant when evaluated in isolation. Both observations are also consistent with precocious ossification rather than delayed ossification. The distribution of the changes seen indicated a lack of specificity of affected ossification which suggests the differences seen are of a random pattern. The number of parameters routinely evaluated makes it highly likely that one or more finding will be seen at a higher level in dosed groups compared with controls. A true developmental effect is only seen when a number of variants or a syndrome of variants is observed, therefore the intergroup difference is considered of no biological significance.
Visceral malformations:
no effects observed
Description (incidence and severity):
Please see attached background material for data tables.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Please see attached background material for results:

Body weight, body weight change and gravid uterine weight,

Body weight change corrected for gravid uterine weight

Number of pregnant and non-pregnant dams

Number of dams with abortions, early deliveries, stillbirths, resorptions, and/or dead foetuses

Pre-and postimplantation loss, number and percent

Mean number and percent of live offspring

Mean foetal/pup body weight by sex and sexes combined

Number and percent of foetuses and litters with malformation and/or variation, description and incidences of malformations and main variations (and/or retardation).

Historical control data

Applicant's summary and conclusion

Conclusions:
The oral administration of Polyphosphoric acids, esters with triethanolamine, sodium salts to pregnant rats by gavage during gestation at dose levels of 100, 500 and 1000 mg/kg bw/day (incorporating a correction factor for water/glycol content (14.47%), did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg bw/day.No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction:

The study was designed to investigate the effects of the test item on adult pregnant females and embryonic and fetal development following repeated administration by gavage to the pregnant female rat during gestation including the period of organogenesis.

 

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods:

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 500, and 1000 mg/kg bw/day (incorporating a correction factor for water/glycol content (14.47%)). A further group of twenty-four time mated females was exposed to the vehicle only to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weight, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality:

There were no unscheduled deaths.

Clinical Observations:

No clinically observable signs of toxicity were evident in treated females.

Body Weight:

No toxicological significant effects in body weight development were detected.

Food Consumption:

No treatment-related effects were detected in food consumption.

Water Consumption:

No adverse effects on water consumption were detected.

Post Mortem Studies:

No macroscopic abnormalities were detected.

Litter Data and Litter Placental and Fetal Weights:

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Fetal Examination:

No treatment-related effects were detected on fetal external findings. No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings in fetuses from females treated with 100, 500 or 1000 mg/kg bw/day.

Conclusion:

The oral administration ofPolyphosphoricacids, esters with triethanolamine, sodium saltsto pregnant rats by gavage during gestation at dose levels of 100, 500 and 1000 mg/kg bw/day (incorporating a correction factor for water/glycol content (14.47%)), did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 1000 mg/kg bw/day.

 

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 1000 mg/kg bw/day.